Cytotoxicity of 2- tert-butyl hydroquinone glutathione conjugates after apical and basolateral exposure of rat renal proximal tubular cell monolayers

Abstract

Confluent monolayers of renal proximal tubular (RPT) cells cultured on porous supports were used to investigate the cytotoxicity induced by glutathione conjugates of 2- tert-butyl-(1,4)-hydroquinone (SG-TBHQ) after apical and basolateral exposure. As judged from lactate dehydrogenase (LDH) leakage, cytotoxicity was observed after basolateral exposure of monolayers to 250 and 500 μ m 2- tert-butyl-5-(glutathion- S-yl)hydroquinone (5SG-TBHQ). In these experiments, LDH leakage was 22.3 ± 1.9 and 32.2 ± 1.9%, respectively. Basolateral exposure of monolayers to 250 and 500 μ m 6SG-TBHQ resulted in LDH leakage of 22.2 ± 2.5 and 30.0 ± 2.7%, respectively. The double conjugate, 2- tert-butyl-3,6-(diglutathione- S-yl)hydroquinone (3,6SG-TBHQ), was not toxic and LDH leakage was about control level (15.0%). Basolaterally located probenecid-sensitive organic anion transporters did not seem to play a part in the cytotoxic effect. However, when RPT cell monolayers were cultured in 24-well tissue culture plates, apical challenge with 250 μ m 5SG-TBHQ induced a cytotoxic effect. In these experiments, LDH leakage was 33.5 ± 0.6%. With these cells, inhibition of apical γ-glutamyltranspeptidase (γGT) activity by acivicin, which was not toxic by itself, decreased 5SG-TBHQ-induced LDH leakage to 19.3 ± 1.2%, whereas 6SG-TBHQ (also 250μ m)-induced LDH leakage was increased to 55.3 ± 1.0%. Co-incubation of RPT cells with SG-TBHQs in the presence of 1.5 m m ascorbic acid (AA) pointed to a pro-oxidant rather than an antioxidant effect of AA. Superoxide dismutase and catalase completely abolished SG-TBHQ-induced cytotoxicity. Since cultured RPT cells lack N-acetylation of cysteine conjugates, the N-deacetylation/ N-acetylation ratio cannot have a vital role in renal toxicity of quinone thioethers in this in vitro system. It seems, therefore, that the cytotoxicity observed is mainly the result of extracellular redox cycling of SG-TBHQs. The lack of toxicity of 6SG-TBHQ after apical exposure could be due to detoxification by γGT-mediated cysteinylglycine- or cysteine-conjugate formation followed by cyclization, as shown for other related quinone glutathione conjugates. The relative importance of the observed effects for the in vivo situation is discussed.